Assignment and Charge Translocation Stoichiometries of the Major Electrogenic Phases in the Reaction of Cytochrome c Oxidase with Dioxygen

Abstract

The reaction of cytochrome c oxidase with dioxygen has been studied by means of time-resolved measurements of electrical membrane potential (DeltaPsi). Microsecond time resolution was achieved by starting with the CO-inhibited enzyme, which was photolyzed after addition of oxygen. The time course of the reaction could be fitted by using a five-step sequential reaction as a model. The first two phases of the reaction, which correspond in time to binding of oxygen followed by formation of the P (peroxy) intermediate, as observed spectroscopically, are not associated with net charge displacement across the membrane. After this lag, DeltaPsi develops in three phases, which correspond in time to the conversion of P to the F (ferryl) intermediate, in a single phase, and conversion of F to O (the fully oxidized enzyme), in two phases. The amplitude of DeltaPsi was approximately equal for the P --> F and F --> O portions of the reaction. When the oxygen reaction is started with incompletely reduced enzyme, it will halt at the P or F state. When the reaction was allowed to proceed to the F state, but no further, only the fast phase of DeltaPsi formation was observed, whereas no DeltaPsi was generated if the reaction was halted at P. This finding places the assignments of phases in the electrometric data on a firmer basis-they are no longer based solely on temporal correspondence with phases in the spectroscopic data. To define the number of charges transferred across the membrane during the reaction, some kind of calibration is needed. For this purpose, another type of reaction-electron transfer following CO photolysis in the absence of oxygen ("backflow")-was studied. Parallel spectroscopic and electrometric measurements showed that the fast electron transfer from the low-spin heme to CuA in the backflow process results in approximately 11 times smaller amplitude of DeltaPsi as compared with DeltaPsi generated in the reaction of the reduced enzyme with oxygen (the polarity is also reversed). If it is assumed that transfer of an electron from the low-spin heme to CuA amounts to movement of a unit charge across half of the membrane dielectric, charge translocation in the reaction of the reduced enzyme with oxygen amounts to approximately 5.5 unit charges-the value predicted if all four protons pumped during the catalytic cycle are translocated during the oxidative part of the reaction.

Plasticity of Proton Pathway Structure and Water Coordination in Cytochrome c Oxidase

“…34. The argonflushed proteoliposome sample contained 5 M valinomycin, 12.5 M (8 nmol) cytochrome c, and 0.5 mM ascorbate.…”

“…electrons and protons) across the membrane plane. In the electrometric measurements with the wild-type CytcO, the voltage changes associated with both the P R 3 F and F 3 O transitions reflect electron transfer from Cu A to heme a, the translocation of pumped protons, and the transfer of substrate protons to the catalytic site, and the ratio of the amplitudes is close to 1 (34). The S197D mutant CytcO does not pump protons, and, as evidenced from the optical measurements, there is at most 10% electron transfer from Cu A to heme a during the P R 3 F transition (see Fig.…”

Discovery of the True Peroxy Intermediate in the Catalytic Cycle of Terminal Oxidases by Real-time Measurement

“…39 and compared with the similar lag phase in CcO. It was established that in CcO the lag can be reasonably modeled as two sequential steps corresponding to the R 3 A and A 3 P R transitions (15). Based on the fact that the lag in cytochrome bd is markedly shorter than that in CcO, the former was fitted as a single step (39).…”

Proton pumping mechanism and catalytic cycle of cytochrome c oxidase: Coulomb pump model with kinetic gating

“…The kinetics of the transition P r to F was studied experimentally in great detail [16,26,27]. However, the protonation state of the ligands in the experimentally formed state is unknown.…”